In this renewal application we describe projects aimed at advancing our understanding of the ubiquitin system. The modified, more general title of the renewal application reflects a considerably expanded program of ubiquitin studies as described in this proposal. Ubiquitin, a 76-residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to a wide variety of protein species, from chromosomal histones to cytoplasmic proteins [1-6]. Earlier studies have suggested that ubiquitin-protein conjugates are preferred substrates for a nonlysosomal, ATP-dependent proteolytic pathway found in reticulocyte extracts [4,6-9]. We recently identified the first mutant in the ubiquitin pathway, a mouse cell cycle mutant ts85 [10], and used it to demonstrate the predominant role of ubiquitin-dependent proteolytic pathway(s) in selective protein degradation [11]. We then established the existence of ubiquitin in the yeast S. cerevisiae and cloned the yeast ubiquitin gene (ub) which was found to encode a polyubiquitin protein precursor [12]. Our major aims in the proposed further studies in both yeast and higher eukaryotes are as follows: 1) Construction and use of the monoubiquitin yeast gene ("unigene"); 2) Chromosomal mapping and determination of the null phenotype of the ub gene; 3) Genetic and biochemical studies on the regulation of ub gene expression and its functional significance; 4) Isolation, characterization and use of ts-mutants in the yeast ub gene and of corresponding extragenic supressors; 5) Use of dominant yeast ts-ub alleles to specifically perturb the ubiquitin system in higher eukaryotes; 6) Detection and both biochemical and genetic characterization of polyubiquitin-processing proteinase(s); 7) Use of lacZ fusions in yeast to address the in vivo functions of ubiquitination; 8) Isolation and characterization of yeast mutants in ATP-dependent proteolysis; 9) Elimination of the putative ubiquitination site in the yeast H2A histone; 10) Use of ubiquitin-specific antibodies as specific inhibitors of the ubiquitin pathway, and in biochemical and histochemical studies on the functions of ubiquitin in the nucleus; 11) Further studies of mouse ts85 cells to address functions of the ubiquitin system in DNA replication and repair; use of the polyoma virus-ts85 system. The results of these studies should contribute to the understanding of selective protein turnover and its relationship to DNA transactions and both normal and malignant patterns of cellular growth.